Abstract: Our research team has been studying Gulf War Veterans with chronic gastrointestinal pain disorders for over a decade (Dunphy et al., 2003). Many Gulf War Veterans developed chronic gastrointestinal pain during their deployment to the Persian Gulf in 1990-91. The pathophysiologic mechanisms of this chronic gastrointestinal pain are not well understood but cause significant morbidity in our Gulf War Veteran population. The mechanisms underlying this chronic gastrointestinal pain disorder in Gulf War Veterans need to be established. In preliminary studies, we have identified unique microRNAs (miRNAs) present in the colon tissue of Gulf War Veterans who returned with chronic gastrointestinal pain. Based on these preliminary findings, our overall hypothesis is: Aberrant expression of specific miRNAs dysregulates downstream miRNA targets (?glutamate & ?opioid receptors) that cause, contribute to, or facilitate chronic gastrointestinal pain in Gulf War Veterans. We have established techniques to determine inter- and intra-cellular roles of miRNAs in the epigenetic regulation of the expression of their down-stream target genes. These methods include: (i) interaction analysis of miRNAs; (ii) transfection of miRNAs into cultures of human colonic epithelial cells; into rat colonic epithelial cells and into rat dorsal root ganglion cells (Aim 3) (iii) in vivo injection of miRNAs in a validated rat model to test for a reduction in visceral hypersensitivity (Greenwood-Van Meerveld et al., 2015; Zhou et al., 2015). Identifying the role of miRNA signaling should: (i) Fill an important gap in our understanding of chronic gastrointestinal pain, especially the dysregulation of nociceptive pathways. (ii) Lead to therapeutic strategies that use small molecules that mimic or inhibit specific miRNAs that target gene expression. This will overcome a critical barrier - lack of effective treatments for our Gulf War Veterans who suffer from chronic gastrointestinal pain disorders and lead to the development of novel, miRNA-based sets of diagnostic markers.